Figure 2. ERK1/2 mediates collagen repression of let-7 in PDAC cells.

A. Panc1 cells were transiently transfected with siRNA against MT1-MMP or control siRNA (50 nmoles) as detailed in Fig. 1D. Cells were allowed to recover overnight and then plated in 3D type I collagen gels (5 mg/ml) for 24 hours, the matrix was dissolved in collagenase (Worthington Biologicals, Lakewood, NJ) and then lysed in m-RIPA buffer. Lysates were immunoblotted for ppERK1/2, ERK/12 and α-tubulin (loading control). Data is representative of 3 separate experiments. B. Panc1 cells were grown on tissue culture plastic or in 3D type I collagen gels (5 mg/ml) for 24 hours and treated with vehicle control (DMSO) or U0126 (10 μM; Cell Signaling, Danvers, MA) for 24 hours. Cells grown in collagen were recovered with collagenase treatment and then lysed in m-RIPA buffer, prior to immunoblotting for ppERK1/2 and α-tubulin (loading control). Data is representative of 4 separate experiments. C. Panc1 cells were grown on plastic or in 3D type I collagen gels (5 mg/ml) for 24 hours and then treated with DMSO, AG1478 (10 μM; Calbiochem, Gibbstown, NJ) or PD153035 (10 μM; Calbiochem, Gibbstown, NJ) for an additional 24 hours. Cells were lysed as before and then immunoblotted for ppERK1/2, ERK2 and α-tubulin (loading control). Data is representative of 3 separate experiments. D. Panc1 cells grown on plastic or in collagen gels were treated with DMSO or U0126 for 24 hours. MicroRNA were isolated using mirVana kit and analyzed for expression of let-7a, let-7d and let-7g by qRT-PCR, with RNU48 as the endogenous control, using gene specific Taqman probes. The data were quantified by the comparative C_T method for relative gene expression from 4 separate experiments. *, p < 0.05; **, p < 0.01 relative to DMSO-treated cells grown in collagen.